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10Ab 10Bb 15

22A 22 Bb 26Ab 26Bb 200

b aAII lines including the control are derived from a single Duboisia clone. bA and B denominate independent regenerants from a single hairy root line.

E. Other Traits of Interest

Apart from modifiying secondary metabolite composition and content, the improvement of agronomic traits such as herbicide resistance, pest and disease resistance, and frost tolerance in Duboisia through genetic modification is also of interest. The most widely used systems for herbicide tolerance are the Roundup Ready and Liberty Link technologies, providing tolerance to the herbicides glyphosate and glufosinate, respectively (47-49). Bacillus thuringiensis endotoxin genes have been used successfully for the improvement of resistance to various insect pests in transgenic crops (47). There are also promising developments in the field of the genetic modification of nematode tolerance (48). Nematodes are very difficult to control chemically, and the nematocides currently available have highly undesirable environmental characteristics (51). Natural plant nematode tolerance genes such as the Mi gene from tomato or the HS1 gene from sugar beet have been identified and cloned. Other approaches rely on the use of proteinase inhibitor genes such as the oryzacystatin gene from rice or the BARNASE/BARSTAR gene system (50). The improvement of frost tolerance via genetic modification is still in its infancy. The detection and subsequent isolation of plant "antifreeze"

Figure 7 (a) Photograph showing two transgenic Duboisia hybrid plants in the greenhouse. The plants have been transformed with the E. coli GUS gene and the NPTII selectable marker gene. No phenotypical difference could be observed in comparison with greenhouse plants of the same clone of the same age. (b) Results of an enzymatic assay demonstrating the activity of the GUS gene in leaves of the two transgenic plants shown in (a). Note the dark (blue) appearance of the leaf pieces (middle and bottom), indicating GUS activity, in contrast to the pale appearance of control leaves (top).

Figure 7 (a) Photograph showing two transgenic Duboisia hybrid plants in the greenhouse. The plants have been transformed with the E. coli GUS gene and the NPTII selectable marker gene. No phenotypical difference could be observed in comparison with greenhouse plants of the same clone of the same age. (b) Results of an enzymatic assay demonstrating the activity of the GUS gene in leaves of the two transgenic plants shown in (a). Note the dark (blue) appearance of the leaf pieces (middle and bottom), indicating GUS activity, in contrast to the pale appearance of control leaves (top).

proteins (52), however, may open up a new route to the improvement of frost tolerance in transgenic plants.

F. Molecular Markers

Molecular markers can be used for genotype identification (genetic fingerprinting), estimation of the genetic diversity of natural populations or breeding stock, and marker-assisted selection of agronomic traits (53). We have established the random amplified polymorphic DNA (RAPD) marker technology (54,55) for application in Duboisia (Fig. 8). Using this technology, we are now able to discriminate and identify our production clones unam-bigously. This ability may serve as a quality control tool during clonal propagation and as a deterrent against theft of our proprietary elite clones. De-

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